Pig nasal mucosa epithelial cell separation culture method

Abstract

The invention is suitable for the technical field of cell separation and culture, and provides a pig nasal mucosa epithelial cell separation and culture method. A mixed enzyme overnight digestion method is adopted, mucous membrane tissue mucus is effectively removed, and more complete digestion is achieved. The pig nasal mucosa epithelial cells with relatively high purity are obtained through a differential attachment technology. And the culture medium is replaced by a complete culture medium containing EGF and vitamin A during first liquid replacement, so that a stable and excellent in-vitro growth environment is provided for epithelial cells. The method not only can rapidly and accurately separate the primary cells, but also ensures the stability and high quality of the culture state of the epithelial cells. According to the method, the blank of porcine nasal mucosa primary cell separation research is filled up, the primary porcine nasal mucosa epithelial cell in-vitro model which is simple and easy to implement and high in repeatability is constructed, and the model can simulate physiological and morphological characteristics of porcine nasal mucosa and can be used for preparing the porcine nasal mucosa epithelial cell in-vitro model. And a reliable in-vitro experimental basis is provided for researches on respiratory tract pathogen infection mechanisms, respiratory system disease drug screening, gene therapy and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of cell separation and culture, and in particular relates to a method for separating and culturing pig nasal mucosal epithelial cells. Background Art

[0002] As the gateway to the respiratory tract, the nasal cavity is the first line of defense against microbial invasion and plays a key role. Nasal epithelial cells are an important component of innate immunity. They are not only a classic physical barrier, but also can prevent the development of inflammation by regulating the secretion of cytokines under the mediation of other harmful substances (viruses, bacteria, and allergens, etc.). Therefore, nasal epithelial cells play an important role in respiratory innate immunity and the secondary cell structure changes and pathological changes caused by them.

[0003] The mucosal immune system (MIS) is an important part of the entire immune network of the body, including exocrine glands such as the respiratory mucosa, gastrointestinal mucosa, reproductive mucosa and conjunctiva, and plays an active and important role in resisting infection. Mucosal immunity is an important part of the pig immune network. More than 50% of lymphoid tissues and 80% of immune cells exist in the mucosa, and the mucosa is the largest immune system of pigs. However, with the continuous development of large-scale pig farming, respiratory diseases in pigs are becoming more and more serious. In view of the complexity of the nasal mucosa, current studies on the pharmacokinetic properties of respiratory drugs mostly rely on in vivo animal models and in vitro tissue specimens, but more suitable in vitro research models are urgently needed to reduce the use of experimental animals in order to comply with the "3R principle". At present, in vitro research models are mainly composed of human nasal epithelial cells or RPMI2650 cell lines, but neither model can meet all relevant requirements. Although the advantages of primary cultured human nasal epithelium are the presence of all physiological cell types, organic structures and similar protein expression patterns. However, its shortcomings cannot be ignored, especially the limited lifespan of human-derived cell materials, the low subculture rate of cells, and the significant differences in cell states caused by individual differences in donors, which in turn affect the formation of barrier properties of cell culture models. Although the use of air-liquid interface (ALI) culture can increase organic properties (such as the expression of mucus and active cilia), the preciousness of human nasal mucosa samples has brought great difficulties and challenges to the establishment of a model of primary human nasal mucosal epithelial cells.

[0004] In this context, pigs, as an excellent model for human disease research, are the only choice for alternative medicine. However, the current infection models used for porcine upper respiratory tract disease research are mainly classic 2D cell lines and related animal models, all of which have many defects, such as limited cell sources, low fidelity and low differentiation ability, long growth cycle, insufficient supply, and high production costs, so it is urgent to explore new pathogen infection models. At present, there is little research on the isolation technology of porcine nasal mucosal epithelial cells at home and abroad, which is mainly limited to the study of the mechanism of disease occurrence and development. In view of this, we urgently need to develop a new and efficient method for isolating porcine nasal mucosal epithelial cells, which can be used as a cell model for in vitro drug transport, disease infection, cell differentiation and other research. To this end, the present invention proposes a method for isolating and culturing porcine nasal mucosal epithelial cells. Summary of the invention

[0005] The purpose of the present invention is to provide a method for isolating and culturing porcine nasal mucosal epithelial cells, aiming to solve the problems raised in the above-mentioned background technology.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for isolating and culturing pig nasal mucosal epithelial cells comprises the following steps:

[0008] Step 1, sampling: separate the pig maxilla, disinfect the tissue material with 75% alcohol, and then remove the pig nasal mucosa within 15 minutes in a sterile environment, wash the removed pig nasal mucosa with 4°C pre-cooled sterile PBS containing 3 antibodies for 3 times, and store the pig nasal mucosa in DMEM / F12 (1:1) culture medium containing 3 antibodies;

[0009] Step 2: Isolation of pig nasal mucosal epithelial cells: Cut the pig nasal mucosal epithelial tissue into 2-3 mm 3 The tissue blocks of different sizes were washed five times with sterile PBS containing 3 antibodies and placed in sterile centrifuge tubes; mixed digestion enzyme solution was added to the tissue blocks for enzymatic digestion, and the specimens were completely immersed in the mixed digestion enzyme solution and digested overnight at 4°C; the next day, the digestion was terminated with preheated DMEM / F12 culture medium containing 10% fetal bovine serum, filtered through a 100μm mesh, and centrifuged at 1000rpm / min at 4°C for 5min to collect cells; 5 times the volume of complete The culture medium was gently pipetted and mixed; filtered through a 70 μm mesh, centrifuged at 1000 rpm / min for 5 min at 4°C to collect the filtered cells, and the obtained cell suspension was transferred to a new cell culture dish and incubated at 37°C for 1 h to remove fibroblasts; the upper cell suspension was collected, centrifuged at 1000 rpm / min for 5 min at 4°C, and 3 times the volume of complete culture medium was added to the cell pellet again, and gently pipetted and mixed;

[0010] Step 3: Culture of porcine nasal mucosal epithelial cells: Count the cells under a microscope, using 5×10 5 The cells were inoculated into a new sterile cell culture dish at a cell density of 1.444 × 10 / ml and cultured in a 37°C, 5% CO2 incubator for 48 hours. The culture medium was then replaced with complete nasal epithelial culture medium and the medium was changed every 2 days. After 8-10 days of culture, the cells grew into a single layer of epithelial cells with a density of 80%-90%.

[0011] Furthermore, the 3 antibodies consist of penicillin, streptomycin and amphotericin B, the concentration of penicillin is 100 U / ml, the concentration of streptomycin is 100 μg / ml, and the concentration of amphotericin B is 0.25 μg / ml.

[0012] Furthermore, the osmotic pressure of the DMEM / F12 culture medium is 280-320 mOsm / kg, and the pH is 7.0-7.4.

[0013] Furthermore, in step 2, the mixed digestive enzyme solution is composed of 0.5 mg / ml collagenase I, 0.5 mg / ml collagenase IV and 0.16 mg / ml hyaluronidase; the volume of the mixed digestive enzyme solution used is 3.5-5 times the volume of the pig nasal mucosal tissue block; the enzymatic digestion time is 16-18 hours; and the enzymatic digestion temperature is 4°C.

[0014] Furthermore, in step 2, the digestion is terminated using DMEM / F12 culture medium preheated to 37° C. containing 10% fetal bovine serum.

[0015] Furthermore, in step 2, the complete culture medium is DMEM / F12 culture medium containing 10% fetal bovine serum.

[0016] Furthermore, in step 3, the culture medium used in the cell culture dish is DMEM / F12 culture medium containing 3 antibodies and 10% fetal bovine serum, which is replaced with complete culture medium after 48 hours of culture. The complete culture medium is DMEM / F12 culture medium containing 3 antibodies, 10% fetal bovine serum, EGF (epidermal growth factor) and vitamin A.

[0017] Furthermore, the concentration of EGF is 25 ng / ml, and the concentration of vitamin A is 1 ug / ml.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The present invention processes porcine nasal mucosal tissue and adopts a mixed enzyme overnight digestion method to effectively remove mucus from the mucosal tissue and achieve more complete digestion. Porcine nasal mucosal epithelial cells with high purity are obtained by differential adhesion technology. And when the medium is replaced for the first time, the culture medium is replaced with a complete medium containing EGF and vitamin A to provide a stable and excellent in vitro growth environment for epithelial cells. This method not only quickly and accurately separates primary cells, but also ensures the stability and high quality of the culture state of epithelial cells.

[0020] 2. The present invention fills the gap in the research on the isolation of primary cells of porcine nasal mucosa, and aims to construct a simple, easy and highly reproducible in vitro model of primary porcine nasal mucosal epithelial cells. This model can simulate the physiological and morphological characteristics of porcine nasal mucosa, and provide a reliable in vitro experimental basis for the study of respiratory pathogen infection mechanisms, respiratory disease drug screening and gene therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a flow chart of the present invention.

[0022] Figure 2 This is a light microscopic observation of the morphological characteristics and growth status of pig nasal mucosal epithelial cells (10×).

[0023] Figure 3 Figure 2 is an immunofluorescence identification image of CK18 in porcine nasal mucosal epithelial cells (20×); (a) is the CK18, DAPI and MERGE images, and (b) is the statistical analysis result showing the cell purity.

[0024] Figure 4 This is an immunofluorescence identification image of ZO-1 in porcine nasal mucosal epithelial cells (10×). DETAILED DESCRIPTION

[0025] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of the present invention. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0026] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0027] like Figure 1 As shown, a method for isolating and culturing porcine nasal mucosal epithelial cells provided by one embodiment of the present invention comprises the following steps:

[0028] Step 1, sampling: separate the pig maxilla, use 75% alcohol to disinfect the tissue material (immerse for 10-15 minutes), then remove the pig nasal mucosa in a sterile environment within 15 minutes, wash the removed pig nasal mucosa with 4°C pre-cooled sterile PBS containing 3 antibodies (penicillin, streptomycin and amphotericin B, concentrations of 100U / ml, 100μg / ml and 0.25μg / ml, respectively), and store the pig nasal mucosa in DMEM / F12 (1:1) culture medium containing 3 antibodies.

[0029] Step 2: Isolation of porcine nasal mucosal epithelial cells: Cut the porcine nasal mucosal epithelial tissue into 2-3 mm pieces using sterile ophthalmic scissors. 3 The tissue blocks of different sizes were washed five times with sterile PBS containing 3 antibodies and then placed in a sterile centrifuge tube; mixed digestion enzyme solution was added to the tissue blocks for enzymatic digestion, so that the specimens were completely immersed in the mixed digestion enzyme solution, wherein the mixed digestion enzyme solution consisted of 0.5 mg / ml collagenase I, 0.5 mg / ml collagenase IV and 0.16 mg / ml hyaluronidase; the volume of the mixed digestion enzyme solution used was 3.5-5 times the volume of the porcine nasal mucosa tissue block, and digestion was carried out overnight at 4°C, which usually took 16-18 hours; the next day, the digestion was terminated with DMEM / F12 culture medium preheated to 37°C containing 10% fetal bovine serum, and filtered through a 100 μm mesh. After that, centrifuge at 1000 rpm / min for 5 min at 4°C to collect the cells; add 5 times the volume of complete culture medium to the cell pellet and mix it gently with a pipette; filter through a 70 μm mesh, centrifuge at 1000 rpm / min for 5 min at 4°C to collect the filtered cells, transfer the obtained cell suspension to a new cell culture dish, and incubate at 37°C for 1 h to remove fibroblasts; collect the upper cell suspension, centrifuge at 1000 rpm / min for 5 min at 4°C, add 3 times the volume of complete culture medium to the cell pellet again, and mix it gently with a pipette;

[0030] In step 2, the complete culture medium is DMEM / F12 culture medium containing 10% fetal bovine serum.

[0031] Step 3: Culture of porcine nasal mucosal epithelial cells: Count the cells under a microscope, using 5×10 5 / ml of cell density, the cells were inoculated into a new sterile cell culture dish, the culture medium used in the cell culture dish was DMEM / F12 culture medium containing 3 antibodies and 10% fetal bovine serum, and after culturing in a 37°C, 5% CO2 incubator for 48 hours, the culture medium was replaced with a complete culture medium, the complete culture medium was DMEM / F12 culture medium containing 3 antibodies, 10% fetal bovine serum, EGF (concentration of 25ng / ml) and vitamin A (1ug / ml), and the medium was replaced once every 2 days; after 8-10 days of culture, the cells grew into a single layer of epithelial cells with a density of 80%-90%.

[0032] As a preferred embodiment of the present invention, the osmotic pressure of the DMEM / F12 culture medium is 280-320 mOsm / kg, and the pH is 7.0-7.4.

[0033] Example 1: Observe the cell growth under an optical microscope. Figure 2 As shown, it was observed that the pig nasal mucosal epithelial cells isolated and cultured by the mixed enzyme overnight digestion method could be seen attached to the wall on the second day, the degree of confluence reached 40%-50% on the fourth day, and the cell confluence reached more than 90% on the tenth day.

[0034] Primary porcine nasal epithelial cells with a cell fusion degree of more than 90% can be passaged. First, discard the original cell culture medium, add 2ml PBS to wash 1-2 times, and remove PBS with a pipette. Add 1-2ml pancreatin (enough to cover the cell surface) along the side wall of the culture dish for digestion for about 5-10min. Under an inverted microscope, the cell morphology is observed to gradually shrink from irregular to round, indicating that the digestion is complete. At this time, add 3ml complete culture medium to terminate the trypsin digestion. Then use a pipette to repeatedly and gently blow the bottom wall of the culture dish or the bottom wall of the culture bottle to detach the cells from the bottom wall. Transfer the culture medium after blowing to a 15ml centrifuge tube and centrifuge at a speed of 1000rpm / min for 5min to obtain a single cell precipitate. After centrifugation, discard the supernatant to obtain a single cell precipitate, resuspend the cell precipitate with 2mL of complete culture medium, divide it into 2 parts and add it to the newly prepared 5ml nasal epithelial cell complete culture medium, and observe the cell density under an optical microscope. Then put it in a constant temperature incubator for culture. After subculture, the cells were observed to adhere to the wall and grow stably after 4 days.

[0035] Example 2: Immunofluorescence identification of isolated cells as epithelial-derived, comprising the following steps:

[0036] The coverslips containing primary cells of porcine nasal epithelial cells were rinsed with PBS for 5 minutes for 3 times, and then fixed with 4% paraformaldehyde for 30 minutes. Permeabilization was performed with 0.1% Triton X-100 for 10 minutes at room temperature; 4% fetal bovine serum was used for blocking for 30 minutes; CK18 antibody (1:300) was incubated at 4°C overnight. Then, PBS was used for 5 minutes, and then 3 times, and FITC-labeled secondary antibody (1:500) was added, and incubated at 37°C for 2 hours; DAPI was used to stain the cell nucleus. After PBS was rinsed for 5 minutes and 3 times, the samples were observed with a confocal microscope, and fluorescence coloration indicated positive.

[0037] The results are as follows Figure 3As shown in the figure, CK18 was used to identify cultured porcine nasal mucosal epithelial cells. Under a fluorescence microscope, CK18 was expressed in the cytoplasm and showed green fluorescence, confirming that the cells were epithelial-derived cells. DAPI was used to stain the cell nucleus in the same field of view. The MERGE image shows that the cells in the field of view were stained with CK18 (green fluorescence) and DAPI (blue fluorescence) at the same time ( Figure 3 If cells show green fluorescence in the MERGE image, it means that these cells express CK18, thus confirming that they are epithelial cells. It can be seen that the purity of nasal mucosal epithelial cells cultured by mixed enzyme overnight digestion method is as high as more than 90% ( Figure 3 (b)).

[0038] Example 3: Immunofluorescence identification of tight junctions of cultured cells, comprising the following steps:

[0039] The coverslip containing primary cells of porcine nasal mucosal epithelial cells cultured for 14 days was rinsed with PBS for 5 minutes three times, and then fixed with 4% paraformaldehyde for 30 minutes. Permeabilization was performed with 0.1% Triton X-100 at room temperature for 10 minutes; blocking was performed with 4% fetal bovine serum for 30 minutes; incubation with ZO-1 antibody (1:500) at 4°C overnight. Then, the samples were rinsed with PBS for 5 minutes, rinsed three times in total, and FITC-labeled secondary antibody (1:500) was added, and incubated at 37°C for 2 hours; DAPI was used to stain the cell nucleus. After rinsing with PBS for 5 minutes, rinsed three times in total, the samples were observed with a confocal microscope, and fluorescence coloration indicated positivity.

[0040] The results are as follows Figure 4 As shown in the figure, ZO-1 was used to identify porcine nasal mucosal epithelial cells cultured for 14 days. Under a fluorescence microscope, ZO-1 was observed to be expressed on the cell membrane with red fluorescence, and the expression level was high, showing uniform and continuous distribution. In the same field of view, DAPI was used to stain the cell nucleus. The MERGE image shows the expression level of ZO-1 in cells in the field of view. These results show that the tight junctions of the porcine nasal mucosal epithelial barrier were good after 14 days of culture.

[0041] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A method for isolating and culturing porcine nasal mucosal epithelial cells, characterized in that: The following steps are involved: Step 1, sampling: separate the pig maxilla, disinfect the tissue material with 75% alcohol, then remove the pig nasal mucosa within 15 minutes in a sterile environment, wash the removed pig nasal mucosa with 4°C pre-cooled sterile PBS containing 3 antibodies for 3 times, and store the pig nasal mucosa in DMEM / F12 culture medium containing 3 antibodies; Step 2: Isolation of pig nasal mucosal epithelial cells: Cut the pig nasal mucosal epithelial tissue into 2-3 mm 3 The tissue blocks of different sizes were washed five times with sterile PBS containing 3 antibodies and placed in sterile centrifuge tubes; mixed digestion enzyme solution was added to the tissue blocks for enzymatic digestion, so that the specimens were completely immersed in the mixed digestion enzyme solution and digested overnight at 4°C; the next day, the digestion was terminated with preheated DMEM / F12 culture medium containing 10% fetal bovine serum, filtered with a 100μm mesh, and centrifuged at 1000rpm / min at 4°C for 5min to collect cells; 5 times the volume of complete culture medium was added to the cell pellet, and gently pipetted and mixed with a pipette; filtered with a 70μm mesh, centrifuged at 1000rpm / min at 4°C for 5min to collect the filtered cells, and the obtained cell suspension was transferred to a new cell culture dish and incubated at 37°C for 1h to remove fibroblasts; Collect the upper cell suspension, centrifuge at 1000 rpm / min for 5 min at 4°C, add 3 times the volume of complete medium to the cell pellet again, and mix gently with a pipette; Step 3: Culture of porcine nasal epithelial cells: Count the cells under a microscope, using 5×10 5 The cells were inoculated into a new sterile cell culture dish at a cell density of 1.444 × 10 / ml and cultured in a 37°C, 5% CO2 incubator for 48 hours. The complete culture medium was then replaced with nasal mucosal epithelial complete culture medium, with the medium changed every 2 days. After 8-10 days of culture, the cells grew into a single layer of epithelial cells with a density of 80%-90%.

2. The method for isolating and culturing porcine nasal mucosal epithelial cells according to claim 1, characterized in that: The 3 antibodies consisted of penicillin, streptomycin and amphotericin B, with the concentration of penicillin being 100 U / ml, the concentration of streptomycin being 100 μg / ml, and the concentration of amphotericin B being 0.25 μg / ml.

3. The method for isolating and culturing porcine nasal mucosal epithelial cells according to claim 1, characterized in that: The osmotic pressure of the DMEM / F12 culture medium is 280-320 mOsm / kg, and the pH is 7.0-7.

4.

4. The method for isolating and culturing porcine nasal mucosal epithelial cells according to claim 1, characterized in that: In step 2, the mixed digestive enzyme solution is composed of 0.5 mg / ml collagenase I, 0.5 mg / ml collagenase IV and 0.16 mg / ml hyaluronidase; the volume of the mixed digestive enzyme solution used is 3.5-5 times the volume of the pig nasal mucosal tissue block; the enzymatic digestion time is 16-18 hours; and the enzymatic digestion temperature is 4°C.

5. The method for isolating and culturing porcine nasal mucosal epithelial cells according to claim 1, characterized in that: In step 2, the digestion is terminated using DMEM / F12 culture medium preheated to 37° C. containing 10% fetal bovine serum.

6. The method for isolating and culturing porcine nasal mucosal epithelial cells according to claim 1, characterized in that: In the step 2, the complete culture medium is DMEM / F12 culture medium containing 10% fetal bovine serum.

7. The method for isolating and culturing porcine nasal mucosal epithelial cells according to claim 1, characterized in that: In step 3, the culture medium used in the cell culture dish is DMEM / F12 culture medium containing 3 antibodies and 10% fetal bovine serum, which is replaced with nasal mucosal epithelial complete culture medium after 48 hours of culture. The complete culture medium is DMEM / F12 culture medium containing 3 antibodies, 10% fetal bovine serum, EGF and vitamin A.

8. The method for isolating and culturing porcine nasal mucosal epithelial cells according to claim 1, characterized in that: In step 3, in the complete culture medium for nasal mucosal epithelium, the concentration of EGF is 25 ng / ml, and the concentration of vitamin A is 1 ug / ml.

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